Development and Validation of a Differentiating Infected from Vaccinated Animals (DIVA) Enzyme-Linked Immunosorbent Assay (ELISA) Strategy for Distinguishing Between Hendra-Infected and Vaccinated Horses.
Abstract: Hendra virus (HeV) is a bat-borne zoonotic agent which can cause a severe and highly fatal disease and can be transferred from animals to humans. It has caused over 100 deaths in horses since it was discovered in 1994. Four out of seven infected humans have died. Since the release of the HeV vaccine (Equivac® HeV Hendra Virus Vaccine for Horses, Zoetis Australia Pty Ltd., Rhodes, NSW 2138) in Australia, there has been an urgent requirement for a serological test for differentiating infected from vaccinated animals (DIVA). All first-line diagnostic serological assays at the Australian Centre for Disease Preparedness (ACDP) incorporate recombinant HeV soluble G glycoprotein (sG) as the antigen, which is also the only immunogen present in the Equivac® HeV vaccine. Problems therefore arose in that antibody testing results were unable to distinguish between prior vaccination or infection with HeV. This study describes the development of a HeV DIVA ELISA strategy using recombinant sG and HeV nucleoprotein (N), paired with specific monoclonal antibodies in a competition ELISA format. The validation of this assay strategy was performed using a positive cohort of 19 serum samples representing post-infection sera, a negative cohort of 1138 serum samples representing horse sera collected pre-vaccine release and a vaccination cohort of 502 serum samples from horses previously vaccinated with Equivac® HeV vaccine. For the sG glycoprotein, the diagnostic sensitivity (DSe) was 100.0% (95% CI: 99.3-100.0%) and diagnostic specificity (DSp) 99.91% (95% CI: 99.5-100.0%), using a percentage inhibition cut-off value of >36, whereas for the N protein, DSe was 100.0% (95% CI: 82.4-100.0%) and DSp 100.0% (95% CI: 99.7-100.0%), using a percentage inhibition cut-off value of >49. Taken together, these results demonstrate that the HeV DIVA ELISA strategy developed here is now an essential and critical component of the testing algorithm for HeV serology testing in Australia.
Publication Date: 2025-02-28 PubMed ID: 40143282PubMed Central: PMC11945769DOI: 10.3390/v17030354Google Scholar: Lookup
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- Journal Article
- Validation Study
Summary
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This research has successfully developed and validated an assay (test) technique capable of distinguishing between horses infected with the deadly Hendra virus and those that have been vaccinated against it – a critical capability because the prior assays couldn’t differentiate between vaccinated and infected animals.
Understanding the Hendra Virus and Its Vaccination
- The Hendra virus (HeV) is a potentially lethal agent transmitted from bats to horses and other animals, and also to humans. It was first identified in 1994 and has since caused over 100 fatalities in horses and four in humans.
- The development of a vaccine known as Equivac HeV has been a game-changer in combating the virus. However, a key challenge has been the lack of a testing mechanism that can distinguish between horses that have been vaccinated and those that have been infected with the virus.
The Problem with Existing Diagnostic Methods
- Current serological assays (blood tests used to detect infection) use a recombinant HeV soluble G glycoprotein (sG) as the antigen. This antigen also happens to be the only immunogen used in the Equivac HeV vaccine.
- Due to this overlap, the previous testing methods could not differentiate between a horse’s immune response caused by actual HeV infection and the response caused by the vaccination.
Development and Validation of the DIVA ELISA Strategy
- A new and enhanced testing strategy was needed to overcome this challenge. This led to the development of the DIVA (Differentiating Infected from Vaccinated Animals) ELISA strategy which uses specific monoclonal antibodies in a competitive ELISA format.
- This strategy uses recombinant sG and HeV nucleoprotein (N) to differentiate between HeV infection and vaccination with Equivac HeV.
- Steps were taken to validate this strategy using serum samples from various cohorts, comprised of post-infection sera, pre-vaccine release horse sera, and samples from horses that were vaccinated with Equivac.
Efficiency and Impact of the DIVA ELISA Strategy
- The validation process revealed that the DIVA ELISA strategy was highly effective at accurately identifying vaccinated and infected horses, with a diagnostic sensitivity of 100.0% and specificity nearing 100% for both analyzed antigenic proteins, sG and N.
- The successful development and validation of the DIVA ELISA strategy thus marks a significant advancement in the testing for the Hendra virus in Australia, now forming a crucial part of the serology testing method for the disease.
Cite This Article
APA
McNabb L, McMahon A, Woube EG, Agnihotri K, Colling A, Broder CC, Kucinskaite-Kodze I, Petraityte-Burneikiene R, Bowden TR, Halpin K.
(2025).
Development and Validation of a Differentiating Infected from Vaccinated Animals (DIVA) Enzyme-Linked Immunosorbent Assay (ELISA) Strategy for Distinguishing Between Hendra-Infected and Vaccinated Horses.
Viruses, 17(3).
https://doi.org/10.3390/v17030354 Publication
Researcher Affiliations
- CSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, Australia.
- CSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, Australia.
- Private Consultant, Melbourne, VIC 3000, Australia.
- CSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, Australia.
- CSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, Australia.
- Department of Microbiology and Immunology, Uniformed Services University, Bethesda, MD 20814, USA.
- Institute of Biotechnology, Life Sciences Centre, Vilnius University, LT-01513 Vilnius, Lithuania.
- Institute of Biotechnology, Life Sciences Centre, Vilnius University, LT-01513 Vilnius, Lithuania.
- CSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, Australia.
- CSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, Australia.
MeSH Terms
- Animals
- Horses
- Henipavirus Infections / veterinary
- Henipavirus Infections / diagnosis
- Henipavirus Infections / immunology
- Henipavirus Infections / prevention & control
- Henipavirus Infections / virology
- Hendra Virus / immunology
- Enzyme-Linked Immunosorbent Assay / methods
- Enzyme-Linked Immunosorbent Assay / veterinary
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horse Diseases / immunology
- Horse Diseases / prevention & control
- Antibodies, Viral / blood
- Vaccination / veterinary
- Viral Vaccines / immunology
- Viral Vaccines / administration & dosage
- Australia
- Sensitivity and Specificity
- Serologic Tests / methods
Conflict of Interest Statement
The authors declare no conflicts of interest.
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